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Notes
Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
>chr6:89651669+89651787 119bp TGTCAAAGGGGCATAGCAGT CCTGGCAATCAGCATTGTA
Mut= 101 bp
Wt= 119 bp
Fam=Mut
Hex=Wt
Sequence
Wt Sequence (deletions in lower case):
TTAAAGGACACTTGTCCTTGGTCTCAGCACAGCTGTTGAGTATATAACCCAGATGCTTTGTTTTGAAAGGAATCCATGGGTGGGCCTTTGGGTTTCAGGTGTCCTCTGAAGGCATCACTGTTTTCCCATCTTAAGTCATTATTTCATAAACGCATGGAGCACTGGGCTTCATGGATGTGAACAGGCAAGCCATGAGCAATGGAGCACTGGAAGGCTTATGCTCTgccaggggccctttgtggtatcttcatatattgttttattccatagatgacggggccagtgttcaggaagtgctgacagaaatcagtaaccagaaaacggtgcccaatatttttgtgaataaagtgcacgtgggtggatgtgaccgaactttccaggtaatgatggtcatcagacacggtcctcaacactgtgtgttcacattgcaatttaatccatgtcaaaggggcatagcagtggaaaggcacaggggctgggtatgggataatcctgaagatgcaagggaaatgagctTTTCCATCAATACCTGAGAAGTTATACAATGCTGATTGCCAGGCACAGGAGGGACATTCTTGGCCTGAAGTCAGAGCAGCTGTGGCCCTCATAGAACTGAGTCTACTACCACTCCTCTATGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGAGTGATCATGAGTCCTGTGAGGGATTCATGGTGGCTCAGGAGCAGCTCATGACTCATGGGG
This mutation is a 292 bp deletion beginning at Chromosome 6 position 89,651,453 bp and ending after 89,651,744 bp (GRCm38/mm10). In addition, there is a 23 bp TTACCAGGCCCTGCCCAAATTTC insertion at the deletion site.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 64680 | TGT CAA AGG GGC ATA GCA GT | Wild type Forward | A | |||
| 64681 | CCT GGC AAT CAG CAT TGT A | Common | A | |||
| 64682 | CCA TGA GCA ATG GAG CAC T | Mutant Forward | A | |||
| 64683 | Fluorophore-1 | CTG AAG ATG CAA GGG AAA TGA | Quencher-1 | WT Probe | ||
| 64684 | Fluorophore-2 | AAG GCT TAT GCT CTT TAC CAG GC | Quencher-2 | MUT Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 64680 | 0.40 uM |
| 64681 | 0.40 uM |
| 64682 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
This is the only strain that uses this protocol.